The present invention is concerned generally with the stimulation of angiogenesis in living tissues and organs; and is particularly directed to the regulation of syndecan-4 cytoplasmic domain phosphorylation within endothelial cells in-situ.
Angiogenesis, by definition, is the formation of new capillaries and blood vessels within living tissues; and is a complex process first recognized in studies of wound healing and then within investigations of experimental tumors. Angiogenesis is thus a dynamic process which involves extracellular matrix remodeling, endothelial cell migration and proliferation, and functional maturation of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo, Trends Cell Biology 6: 454-456 (1996)]. Clearly, in normal living subjects, the process of angiogenesis is a normal host response to injury; and as such, is an integral part of the host body""s homeostatic mechanisms.
It will be noted and appreciated, however, that whereas angiogenesis represents an important component part of tissue response to ischemia, or tissue wounding, or tumor-initiated neovascularization, relatively little new blood vessel formation or growth takes place in most living tissues and organs in mature adults (such as the myocardium of the living heart) [Folkman, J. and Y. Shing, J. Biol. Chem. 267: 10931-10934 (1992); Folkman, J., Nat. Med. 1: 27-31 (1995); Ware, J. A. and M. Simons, Nature Med. 3: 158-164 (1997)]. Moreover, although regulation of an angiogenetic response in-vivo is a critical part of normal and pathological homeostasis, little is presently known about the control mechanisms for this process.
A number of different growth factors and growth factor receptors have been found to be involved in the process of stimulation and maintenance of angiogenetic responses. In addition, a number of extracellular matrix components and cell membrane-associated proteins are thought to be involved in the control mechanisms of angiogenesis. Such proteins include SPARC [Sage et al., J. Cell Biol. 109: 341-356 (1989); Motamed, K. and E. H. Sage, Kidney Int. 51: 1383-1387 (1997)]; thrombospondin 1 and 2 respectively [Folkman, J., Nat. Med. 1: 27-31 (1995); Kyriakides et al., J. Cell Biol. 140: 419-430 (1998)]; and integrins xcex1vxcex25 and xcex1vxcex23 [Brooks et al., Science 264: 569-571 (1994); Friedlander et al., Science 270: 1500-1502 (1995)]. However, it is now recognized that a major role is played by heparan-binding growth factors such as basic fibrocyte growth factor (bFGF) and vascular endothelial growth factor (VEGF); and thus the regulation of angiogenesis involves the extracellular heparan sulfate matrix and the core proteins at the surface of endothelial cells.
While growth factor signalling generally occurs through specific high-affinity receptors, several growth factors are now known to interact with adjacent, membrane-anchored, proteoglycan co-receptors. In particular, bFGF requires binding to a specific sequence of sulfated polysaccharides in the extracellular heparan sulfate glycosaminoglycan (GAG) chain [Turnbull et al., J. Biol. Chem. 267: 10337-10341 (1992)] in order to bind to its high-affinity receptor on the cell surface and to exert its effect on the target cells [Olwin, B. B., and A. Rapraeger, J. Cell Biol. 118: 631-639 (1992); Rapraeger et al., Science 252: 1705-1708 (1991)]. The current picture of the role of heparan sulfate in the binding mechanism of bFGF involves dimerization of the growth factor as well as direct heparan sulfate binding to the high-affinity receptor [Brickman et al., J. Biol. Chem. 270: 24941-24948 (1995); Kan et al., Science 259: 1918-1921 (1993)]. Together, these events lead to receptor multimerization and to tyrosine trans-phosphorylation of adjacent FGF receptor cytoplasmic tails, followed by phosphorylation of other downstream substrates [Krufka et al., Biochemistry 35: 11131-11141 (1996); van der Geer et al., Annu. Rev. Cell Biol. 10: 251-337 (1994)].
Research investigations have shown that heparan sulfate core proteins or proteoglycans mediate both heparin-binding growth factors and receptor interaction at the cell surface; and that accumulation and storage of such growth factors within the extracellular matrix proper typically occurs [Vlodavsky et al., Clin. Exp. Metastasis 10: 65 (1992); Olwin, B. B. and A. Rapraeger, J. Cell Biol. 118: 631-639 (1992); Rapraeger, A. C., Curr. Opin. Cell Biol. 5: 844-853 (1993)]. The presence of heparin or heparan sulfate is thus required for bFGF-dependent activation of cell growth in-vitro [Yayon et al., Cell 64: 841-848 (1991); Rapraeger et al., Science 252: 1705-1708 (1991)]; and the removal of heparan sulfate chains from the cell surface and extracellular matrix by enzymatic digestion greatly impairs bFGF activity and inhibits neovascularization in-vivo [Sasisekharan et al., Proc. Natl. Acad. Sci. USA 91: 1524-1528 (1994)]. Ample scientific evidence now exists which demonstrates that any meaningful alteration of heparan sulfate (HS) chain composition on the cell surface or within the extracellular matrix (which can be initiated by means of an altered synthesis, or a degradation, or a substantive modification of glycosaminoglycan chains) can meaningful affect the intracellular signaling cascade initiated by the growth factor. The importance of heparan sulfate in growth factor-dependent signaling has become well recognized in this field.
Heparan sulfate (HS) chains on the cell surface and within the extracellular matrix are present via a binding to a specific category of proteins commonly referred to as xe2x80x9cproteoglycansxe2x80x9d. This category is constituted of several classes of core proteins, each of which serve as acceptors for a different type of glycosaminoglycan (GAG) chains. The GAGs are linear co-polymers of N-acetyl-D-glycosamine [binding heparan sulfate] or N-acetyl-D-galactosamine [binding chondroitin sulfate (CS) chains] and aoidic sugars which are attached to these core proteins via a linking tetrasaccharide moiety. Three major classes of HS-carrying core proteins are present in living endothelial cells: cell membrane-spanning syndecans, GPI-linked glypicans, and a secreted perlecan core protein [Rosenberg et al., J. Clin. Invest. 99: 2062-2070 (1997)]. While the perlecan and glypican classes carry and bear HS chains almost exclusively, the syndecan core proteins are capable of carrying both HS and CS chains extracellularly. The appearance of specific glycosaminoglycan chains (such as HS and/or CS) extracellularly on protein cores is regulated both by the structure of the particular core protein as well as via the function of the Golgi apparatus intracellularly in a cell-type specific manner [Shworak et al., J. Biol. Chem. 269: 21204-21214 (1994)].
Today, it is recognized that the syndecan class is composed of four closely related family proteins (syndecan-1,-2,-3 and -4 respectively) coded for by four different genes in-vivo. Syndecans-1 and -4 are the most widely studied members of this class and show expression in a variety of different cell types including epithelial, endothelial, and vascular smooth muscle cells, although expression in quiescent tissues is at a fairly low level [Bernfield et al., Annu. Rev. Cell Biol. 8: 365-393 (1992); Kim et al., Mol. Biol. Cell 5: 797-805 (1994)]. Syndecan-2 (also known as fibroglycan) is expressed at high levels in cultured lung and skin fibroblasts, although immunocytochemically this core protein is barely detectable in most adult tissues. However, syndecan-3 (also known as N-syndecan) demonstrates a much more limited pattern of expression, being largely restricted to peripheral nerves and central nervous system tissues (although high levels of expression are shown in the neonatal heart) [Carey et al., J. Cell Biol. 117: 191-201(1992)]. All four members of the syndecan class are capable of carrying both HS and CS chains extracellularly, although most of syndecan-associated biological effects (including regulation of blood coagulation, cell adhesion, and signal transduction) are largely thought to be due to the presence of HS chains capable of binding growth factors, or cell adhesion receptors and other biologically active molecules [Rosenberg et al., J. Clin. Invest. 99: 2062-2070 (1997)].
Syndecan-1 expression has been also observed during development suggesting a potential role in the epithelial organization of the embryonic ectoderm and in differential axial patterning of the embryonic mesoderm, as well as in cell differentiation [Sutherland et al., Development 113: 339-351 (1991); Trautman et al., Development 111: 213-220 (1991)]. Also, mesenchymal cell growth during tooth organogenesis is associated with transient induction of syndecan-1 gene expression [Vainio et al., Dev. Biol. 147: 322-333 (1991)]. Furthermore, in adult living tissues, expression of syndecan-1 and syndecan-4 proteoglycans substantially increases within arterial smooth muscle cells after balloon catheter injury [Nikkari et al., Am. J. Pathol. 144: 1348-1356 (1994)]; in healing skin wounds [Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994)]; and in the heart following myocardial infarction [Li et al., Circ. Res. 81: 785-796 (1997)]. In the latter instances, the presence of blood-derived macrophages appears necessary for the induction of syndecan-1 and -4 gene expression.
Presently, however, the effects of changes in syndecan expression on cell behavior are not well understood. For example, this core protein is believed to mediate bFGF binding and cell activity. Overexpression of syndecan-1 in 3T3 cells led to inhibition of bFGF-induced growth [Mali et al., J. Biol. Chem. 268: 24215-24222 (1993)]; while in 293T cells, overexpression of syndecan-1 augmented serum-dependent growth [Numa et al., Cancer Res. 55: 4676-4680 (1995)]. Furthermore, syndecan-1 overexpression showed increased inter-cellular adhesion in lymphoid cells [Lebakken et al., J. Cell Biol. 132: 1209-1221 (1996)] while also decreasing the ability of B-lymphocytes to invade collagen gels [Libersbach, B. F. and R. D. Sanderson, J. Biol. Chem. 269: 20013-20019(1994)]. These ostensibly contradictory findings have merely added to the uncertainty and the disparity of knowledge regarding the effects of syndecan expression.
In addition, although there are significant differences between the sequences of their ectoplasmic domains, the four syndecans share a highly conserved cytoplasmic tail containing four invariant tyrosines and one invariant serine [Kojima et al., J. Biol. Chem. 267: 4870-4877 (1992)]. This degree of conservation may reflect functional similarities between cytoplasmic tails of all the syndecans. However, unlike the well established involvement of the ectoplasmic domain in growth factor binding through the GAG chains, there is still no consensus regarding the function of the cytoplasmic tail. Several reports [Carey et al., J. Cell Biol. 124: 161-170 (1994); Carey et al., Exp. Cell Res. 214: 12-21 (1994)] point to transient association of the cytoplasmic tail of syndecan-1 to the actin cytoskeleton which seems to be highly dependent on the presence of one of the four conserved tyrosines [Carey et al., J. Biol. Chem. 271: 15253-15260 (1996)].
It is recognized also that the 18-amino acid-long cytoplasmic tail of syndecan-4 is the least homologous to the other three syndecans, containing a unique nine-residue sequence (RMKKKDEGSYDLGKKPIYKKAPTNEFYA)(SEQ I.D. NO: 1). Syndecan-4 is incorporated into focal adhesions of fibroblasts in a PKC-dependent manner [Baciu, P. C. and P. F. Goetinck, Mol. Biol. Cell 6: 1503-1513 (1995)]; and its cytoplasmic tail appears to bind and activate PKCxcex1 [Oh et al., J. Biol. Chem. 272: 8133-8136 (1997)]. These capacities are special to the cytoplasmic tail of syndecan-4 and not shared by the other syndecans, because they are mediated through oligomerization of its unique nine-residue sequence [Oh et al., J. Biol. Chem. 272: 11805-11811 (1997)].
Also, the presence of the five conserved phosphorylatable residues in the cytoplasmic tails of all the syndecans has been noted. However, although in-vitro phosphorylation by calcium-dependent PKC of serine residues in partial or complete synthetic cytoplasmic tails was reported for syndecan-2 and syndecan-3, it could not be produced for syndecan-1 or syndecan-4 [Prasthofer et al., Biochem. Mol. Biol. Int. 36: 793-802 (1995); Oh et al., Arch. Biochem. Bio Phys. 344: 67-74 (1997)]. Serine phosphorylation in situ was detected in syndecan-2 of carcinoma cells cultured in the presence of serum [Itano et al., Biochem. J. 325: 925-930 (1996)]. This phosphorylation was attributed to the serine residue in the cytoplasmic tail of syndecan-2, contained within a sequence that conforms to a phosphorylation motif of cAMP and cGMP-dependent kinases. In situ phosphorylation of the cytoplasmic tail of syndecan-1 was produced in mammary gland cells by treatment with orthoyanadate or pervanadate, both of which inhibit tyrosine phosphatase [Reiland et al., Biochem. J. 319: 39-47 (1996)]. Accordingly, this treatment resulted predominantly in tyrosine phosphorylation, although a lesser degree of serine phosphorylation was also detected. One of the four tyrosines in the cytoplasmic tail of syndecan-1 is contained within a tyrosine kinase phosphorylation motif [Gould et al., Proc. Natl. Acad. Sci. USA 89: 3271-3275 (1992)] conserved in all the syndecans and may at least partially account for the orthovanadate and pervanadate-produced phosphorylation.
In sum therefore, it is evident that the quantity and quality of knowledge presently available regarding glycoseaminoglycan (GAG) binding core proteins is factually incomplete, often presumptive, and in some instance apparently contradictory. Clearly the role of specific proteoglycans, and particularly syndecans, as mediators under various conditions is recognized; nevertheless, the mechanisms of action and the functional activity of the various individual syndecan core proteins remains yet to be elucidated. Thus, while the role of proteoglycans generally is known to relate in some manner to angiogenesis, there is no evidence or data as yet which establishes the true functional action of specific proteoglycans nor which provides a means for using specific proteoglycans to stimulate angiogenesis in-situ.
The present invention is comprised of related alternatives and has multiple aspects. One aspect provides a first method for stimulating angiogenesis within various tissues and organs in-situ, said method comprising:
identifying a viable endothelial cell in-situ as a target, said targeted endothelial cell bearing a plurality of transmembrane syndecan-4 proteoglycans positioned at and through the cell surface wherein the 183rd amino acid residue present within the intracellular cytoplasmic domain of said syndecan-4 proteoglycan is a serine residue;
administering to said targeted endothelial cell on at least one occasion a predetermined amount of an inhibitor of Protein Kinase C xcex4 (delta) isoenzyme activity such that said 183rd serine residue within the cytoplasmic domain of at least some of said syndecan-4 proteoglycans is present in a non-phosphorylated state; and
allowing said 183rd serine residue within the cytoplasmic domain of said syndecan-4 proteoglycans to continue to be present in a non-phosphorylated state, whereby a stimulation of angiogenesis in-situ results. Another aspect provides a related, but alternative method for stimulating angiogenesis within viable cells, tissues, and organs in-situ, said alternative method comprising:
identifying a viable endothelial cell in-situ as a target, said targeted endothelial cell bearing a plurality of transmembrane syndecan-4 proteoglycans positioned at and through the cell surface wherein the 183rd amino acid residue present within the intracellular cytoplasmic domain of said syndecan-4 proteoglycan is a serine residue;
administering to said targeted endothelial cell on at least one occasion a predetermined amount of a composition able to increase Protein Kinase C xcex1 (alpha) isoenzyme activity intracellularly such that said 183rd serine residue within the cytoplasmic domain of at least some of said syndecan-4 proteoglycans is present in an non-phosphorylated state in-situ; and
allowing said 183rd serine residue within the cytoplasmic domain of said syndecan-4 proteoglycans to continue to be present in a non-phosphorylated state, whereby a stimulation of angiogenesis in-situ results.
A different aspects provides another related, but alternative method for stimulating angiogenesis within viable cells, tissues, and organs in-situ, said alternative method comprising:
identifying a viable endothelial cell in-situ as a target, said targeted endothelial cell bearing a plurality of transmembrane syndecan-4 proteoglycans positioned at and through the cell surface wherein the 183rd amino acid residue present within the intracellular cytoplasmic domain of said syndecan-4 proteoglycan is a serine residue;
administering to said targeted endothelial cell on at least one occasion a predetermined amount of an substance able to activate at least one enzyme selected from the group consisting of protein phosphatases 1 and 2A such that said 183rd serine residue within the cytoplasmic domain of at least some of said syndecan-4 proteoglycans is present in an non-phosphorylated state; and
allowing said 183rd serine residue within the cytoplasmic domain of said syndecan-4 proteoglycans to continue to be present in a non-phosphorylated state, whereby a stimulation of angiogenesis in-situ results.